LOCUS       sequence-214594-       13381 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Piggyac transposon containing tamoxifen inducible Cas9 and a gRNA 
            cassette..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 13381)
  TITLE     Chen lab CRISPR plasmids
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 13381)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: 
            "Unpublished"
COMMENT     Sequence Label: pPB-US-ECasE
FEATURES             Location/Qualifiers
     source          1..13381
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        complement(1..105)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(131..586)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(268..289)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     480..499
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     713..735
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     727..744
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     728..744
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     repeat_region   874..936
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     promoter        1423..1671
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1423..1443
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     1594..1613
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        1690..1765
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        1857..3035
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          2088..3026
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     2983..3003
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     promoter        3060..3107
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     regulatory      3132..3141
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3141..4076
                     /codon_start=1
                     /product="mutated ligand-binding domain of the human 
                     estrogen receptor (Feil et al., 1997)"
                     /label=ERT2
                     /translation="AGDMRAANLWPSPLMIKRSKKNSLALSLTADQMVSALLDAEPPIL
                     YSEYDPTRPFSEASMMGLLTNLADRELVHMINWAKRVPGFVDLTLHDQVHLLECAWLEI
                     LMIGLVWRSMEHPVKLLFAPNLLLDRNQGKCVEGMVEIFDMLLATSSRFRMMNLQGEEF
                     VCLKSIILLNSGVYTFLSSTLKSLEEKDHIHRVLDKITDTLIHLMAKAGLTLQQQHQRL
                     AQLLLILSHIRHMSNKGMEHLYSMKCKNVVPLYDLLLEAADAHRLHAPTSRGGASVEET
                     DQSHLATAGSTSSHSLQKYYITGEAEGFPAT"
     CDS             4089..8189
                     /codon_start=1
                     /product="Cas9 (Csn1) endonuclease from the Streptococcus 
                     pyogenes Type II CRISPR/Cas system"
                     /label=Cas9
                     /note="generates RNA-guided double strand breaks in DNA"
                     /translation="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIWKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             8196..9131
                     /codon_start=1
                     /product="mutated ligand-binding domain of the human 
                     estrogen receptor (Feil et al., 1997)"
                     /label=ERT2
                     /translation="AGDMRAANLWPSPLMIKRSKKNSLALSLTADQMVSALLDAEPPIL
                     YSEYDPTRPFSEASMMGLLTNLADRELVHMINWAKRVPGFVDLTLHDQVHLLECAWLEI
                     LMIGLVWRSMEHPVKLLFAPNLLLDRNQGKCVEGMVEIFDMLLATSSRFRMMNLQGEEF
                     VCLKSIILLNSGVYTFLSSTLKSLEEKDHIHRVLDKITDTLIHLMAKAGLTLQQQHQRL
                     AQLLLILSHIRHMSNKGMEHLYSMKCKNVVPLYDLLLEAADAHRLHAPTSRGGASVEET
                     DQSHLATAGSTSSHSLQKYYITGEAEGFPAT"
     CDS             9144..9197
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             9198..9794
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APHNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(9198..9217)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     complement(9885..9905)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     polyA_signal    9951..10072
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(9988..10007)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     10042..10061
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     repeat_region   10835..10869
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(11360..11376)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(11360..11376)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(11373..11395)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    11384..11400
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(11408..11414,11415..11432,11433..11438))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    11453..11474
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(11591..11608)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(11762..12350)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(11842..11861)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(12521..13312,13313..13381))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     13144..13163
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata
       61 catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa
      121 agtgccacct aaattgtaag cgttaatatt ttgttaaaat tcgcgttaaa tttttgttaa
      181 atcagctcat tttttaacca ataggccgaa atcggcaaaa tcccttataa atcaaaagaa
      241 tagaccgaga tagggttgag tgttgttcca gtttggaaca agagtccact attaaagaac
      301 gtggactcca acgtcaaagg gcgaaaaacc gtctatcagg gcgatggccc actacgtgaa
      361 ccatcaccct aatcaagttt tttggggtcg aggtgccgta aagcactaaa tcggaaccct
      421 aaagggagcc cccgatttag agcttgacgg ggaaagccgg cgaacgtggc gagaaaggaa
      481 gggaagaaag cgaaaggagc gggcgctagg gcgctggcaa gtgtagcggt cacgctgcgc
      541 gtaaccacca cacccgccgc gcttaatgcg ccgctacagg gcgcgtccca ttcgccattc
      601 aggctgcgca actgttggga agggcgatcg gtgcgggcct cttcgctatt acgccagctg
      661 gcgaaagggg gatgtgctgc aaggcgatta agttgggtaa cgccagggtt ttcccagtca
      721 cgacgttgta aaacgacggc cagtgagcgc gcctcgttca ttcacgtttt tgaacccgtg
      781 gaggacgggc agactcgcgg tgcaaatgtg ttttacagcg tgatggagca gatgaagatg
      841 ctcgacacgc tgcagaacac gcagctagat taaccctaga aagataatca tattgtgacg
      901 tacgttaaag ataatcatgt gtaaaattga cgcatgtgtt ttatcggtct gtatatcgag
      961 gtttatttat taatttgaat agatattaag ttttattata tttacactta catactaata
     1021 ataaattcaa caaacaattt atttatgttt atttatttat taaaaaaaac aaaaactcaa
     1081 aatttcttct ataaagtaac aaaactttta tgagggacag ccccccccca aagcccccag
     1141 ggatgtaatt acgtccctcc cccgctaggg ggcagcagcg agccgcccgg ggctccgctc
     1201 cggtccggcg ctccccccgc atccccgagc cggcagcgtg cggggacagc ccgggcacgg
     1261 ggaaggtggc acgggatcgc tttcctctga acgcttctcg ctgctctttg agcctgcaga
     1321 cacctggggg gatacgggga aaaggcctcc acggccagac tagaggtacc aacgcggcct
     1381 ttttacggtt cctggccttt tgctggcctt ttgctcacat gtgagggcct atttcccatg
     1441 attccttcat atttgcatat acgatacaag gctgttagag agataattgg aattaatttg
     1501 actgtaaaca caaagatatt agtacaaaat acgtgacgta gaaagtaata atttcttggg
     1561 tagtttgcag ttttaaaatt atgttttaaa atggactatc atatgcttac cgtaacttga
     1621 aagtatttcg atttcttggc tttatatatc ttgtggaaag gacgaaacac cgggtcttcg
     1681 agaagacctg ttttagagct agaaatagca agttaaaata aggctagtcc gttatcaact
     1741 tgaaaaagtg gcaccgagtc ggtgcttttt tgttttagag ctagaaatag caagttaaaa
     1801 taaggctagt ccgtttttag cgctcgagct tctaggtctt gaaaggagtg ggaattggct
     1861 ccggtgcccg tcagtgggca gagcgcacat cgcccacagt ccccgagaag ttggggggag
     1921 gggtcggcaa ttgaaccggt gcctagagaa ggtggcgcgg ggtaaactgg gaaagtgatg
     1981 tcgtgtactg gctccgcctt tttcccgagg gtgggggaga accgtatata agtgcagtag
     2041 tcgccgtgaa cgttcttttt cgcaacgggt ttgccgccag aacacaggta agtgccgtgt
     2101 gtggttcccg cgggcctggc ctctttacgg gttatggccc ttgcgtgcct tgaattactt
     2161 ccacctggct gcagtacgtg attcttgatc ccgagcttcg ggttggaagt gggtgggaga
     2221 gttcgaggcc ttgcgcttaa ggagcccctt cgcctcgtgc ttgagttgag gcctggcctg
     2281 ggcgctgggg ccgccgcgtg cgaatctggt ggcaccttcg cgcctgtctc gctgctttcg
     2341 ataagtctct agccatttaa aatttttgat gacctgctgc gacgcttttt ttctggcaag
     2401 atagtcttgt aaatgcgggc caagatctgc acactggtat ttcggttttt ggggccgcgg
     2461 gcggcgacgg ggcccgtgcg tcccagcgca catgttcggc gaggcggggc ctgcgagcgc
     2521 ggccaccgag aatcggacgg gggtagtctc aagctggccg gcctgctctg gtgcctggcc
     2581 tcgcgccgcc gtgtatcgcc ccgccctggg cggcaaggct ggcccggtcg gcaccagttg
     2641 cgtgagcgga aagatggccg cttcccggcc ctgctgcagg gagctcaaaa tggaggacgc
     2701 ggcgctcggg agagcgggcg ggtgagtcac ccacacaaag gaaaagggcc tttccgtcct
     2761 cagccgtcgc ttcatgtgac tccacggagt accgggcgcc gtccaggcac ctcgattagt
     2821 tctcgagctt ttggagtacg tcgtctttag gttgggggga ggggttttat gcgatggagt
     2881 ttccccacac tgagtgggtg gagactgaag ttaggccagc ttggcacttg atgtaattct
     2941 ccttggaatt tgcccttttt gagtttggat cttggttcat tctcaagcct cagacagtgg
     3001 ttcaaagttt ttttcttcca tttcaggtgt cgtgaggaat tagcttggta ccggaccgtg
     3061 ttgacaatta atcatcggca tagtatatcg gcatagtata atacgacaag gtgaggaact
     3121 aaaccggatc cgccaccatg gctggagaca tgagagctgc caacctttgg ccaagcccgc
     3181 tcatgatcaa acgctctaag aagaacagcc tggccttgtc cctgacggcc gaccagatgg
     3241 tcagtgcctt gttggatgct gagcccccca tactctattc cgagtatgat cctaccagac
     3301 ccttcagtga agcttcgatg atgggcttac tgaccaacct ggcagacagg gagctggttc
     3361 acatgatcaa ctgggcgaag agggtgccag gctttgtgga tttgaccctc catgatcagg
     3421 tccaccttct agaatgtgcc tggctagaga tcctgatgat tggtctcgtc tggcgctcca
     3481 tggagcaccc agtgaagcta ctgtttgctc ctaacttgct cttggacagg aaccagggaa
     3541 aatgtgtaga gggcatggtg gagatcttcg acatgctgct ggctacatca tctcggttcc
     3601 gcatgatgaa tctgcaggga gaggagtttg tgtgcctcaa atctattatt ttgcttaatt
     3661 ctggagtgta cacatttctg tccagcaccc tgaagtctct ggaagagaag gaccatatcc
     3721 accgagtcct ggacaagatc acagacactt tgatccacct gatggccaag gcaggcctga
     3781 ccctgcagca gcagcaccag cggctggccc agctcctcct catcctctcc cacatcaggc
     3841 acatgagtaa caaaggcatg gagcatctgt acagcatgaa gtgcaagaac gtggtgcccc
     3901 tctatgacct gctgctggag gcggcggacg cccaccgcct acatgcgccc actagccgtg
     3961 gaggggcatc cgtggaggag acggaccaaa gccacttggc cactgcgggc tctacttcat
     4021 cgcattcctt gcaaaagtat tacatcacgg gggaggcaga gggtttccct gccacagctt
     4081 accggtttga caagaagtac agcatcggcc tggacatcgg caccaactct gtgggctggg
     4141 ccgtgatcac cgacgagtac aaggtgccca gcaagaaatt caaggtgctg ggcaacaccg
     4201 accggcacag catcaagaag aacctgatcg gagccctgct gttcgacagc ggcgaaacag
     4261 ccgaggccac ccggctgaag agaaccgcca gaagaagata caccagacgg aagaaccgga
     4321 tctgctatct gcaagagatc ttcagcaacg agatggccaa ggtggacgac agcttcttcc
     4381 acagactgga agagtccttc ctggtggaag aggataagaa gcacgagcgg caccccatct
     4441 tcggcaacat cgtggacgag gtggcctacc acgagaagta ccccaccatc taccacctga
     4501 gaaagaaact ggtggacagc accgacaagg ccgacctgcg gctgatctat ctggccctgg
     4561 cccacatgat caagttccgg ggccacttcc tgatcgaggg cgacctgaac cccgacaaca
     4621 gcgacgtgga caagctgttc atccagctgg tgcagaccta caaccagctg ttcgaggaaa
     4681 accccatcaa cgccagcggc gtggacgcca aggccatcct gtctgccaga ctgagcaaga
     4741 gcagacggct ggaaaatctg atcgcccagc tgcccggcga gaagaagaat ggcctgttcg
     4801 gaaacctgat tgccctgagc ctgggcctga cccccaactt caagagcaac ttcgacctgg
     4861 ccgaggatgc caaactgcag ctgagcaagg acacctacga cgacgacctg gacaacctgc
     4921 tggcccagat cggcgaccag tacgccgacc tgtttctggc cgccaagaac ctgtccgacg
     4981 ccatcctgct gagcgacatc ctgagagtga acaccgagat caccaaggcc cccctgagcg
     5041 cctctatgat caagagatac gacgagcacc accaggacct gaccctgctg aaagctctcg
     5101 tgcggcagca gctgcctgag aagtacaaag agattttctt cgaccagagc aagaacggct
     5161 acgccggcta cattgacggc ggagccagcc aggaagagtt ctacaagttc atcaagccca
     5221 tcctggaaaa gatggacggc accgaggaac tgctcgtgaa gctgaacaga gaggacctgc
     5281 tgcggaagca gcggaccttc gacaacggca gcatccccca ccagatccac ctgggagagc
     5341 tgcacgccat tctgcggcgg caggaagatt tttacccatt cctgaaggac aaccgggaaa
     5401 agatcgagaa gatcctgacc ttccgcatcc cctactacgt gggccctctg gccaggggaa
     5461 acagcagatt cgcctggatg accagaaaga gcgaggaaac catcaccccc tggaacttcg
     5521 aggaagtggt ggacaagggc gcttccgccc agagcttcat cgagcggatg accaacttcg
     5581 ataagaacct gcccaacgag aaggtgctgc ccaagcacag cctgctgtac gagtacttca
     5641 ccgtgtataa cgagctgacc aaagtgaaat acgtgaccga gggaatgaga aagcccgcct
     5701 tcctgagcgg cgagcagaaa aaggccatcg tggacctgct gttcaagacc aaccggaaag
     5761 tgaccgtgaa gcagctgaaa gaggactact tcaagaaaat cgagtgcttc gactccgtgg
     5821 aaatctccgg cgtggaagat cggttcaacg cctccctggg cacataccac gatctgctga
     5881 aaattatcaa ggacaaggac ttcctggaca atgaggaaaa cgaggacatt ctggaagata
     5941 tcgtgctgac cctgacactg tttgaggaca gagagatgat cgaggaacgg ctgaaaacct
     6001 atgcccacct gttcgacgac aaagtgatga agcagctgaa gcggcggaga tacaccggct
     6061 ggggcaggct gagccggaag ctgatcaacg gcatccggga caagcagtcc ggcaagacaa
     6121 tcctggattt cctgaagtcc gacggcttcg ccaacagaaa cttcatgcag ctgatccacg
     6181 acgacagcct gacctttaaa gaggacatcc agaaagccca ggtgtccggc cagggcgata
     6241 gcctgcacga gcacattgcc aatctggccg gcagccccgc cattaagaag ggcatcctgc
     6301 agacagtgaa ggtggtggac gagctcgtga aagtgatggg ccggcacaag cccgagaaca
     6361 tcgtgatcga aatggccaga gagaaccaga ccacccagaa gggacagaag aacagccgcg
     6421 agagaatgaa gcggatcgaa gagggcatca aagagctggg cagccagatc ctgaaagaac
     6481 accccgtgga aaacacccag ctgcagaacg agaagctgta cctgtactac ctgcagaatg
     6541 ggcgggatat gtacgtggac caggaactgg acatcaaccg gctgtccgac tacgatgtgg
     6601 accatatcgt gcctcagagc tttctgaagg acgactccat cgacaacaag gtgctgacca
     6661 gaagcgacaa gaaccggggc aagagcgaca acgtgccctc cgaagaggtc gtgaagaaga
     6721 tgaagaacta ctggcggcag ctgctgaacg ccaagctgat tacccagaga aagttcgaca
     6781 atctgaccaa ggccgagaga ggcggcctga gcgaactgga taaggccggc ttcatcaaga
     6841 gacagctggt ggaaacccgg cagatcacaa agcacgtggc acagatcctg gactcccgga
     6901 tgaacactaa gtacgacgag aatgacaagc tgatccggga agtgaaagtg atcaccctga
     6961 agtccaagct ggtgtccgat ttccggaagg atttccagtt ttacaaagtg cgcgagatca
     7021 acaactacca ccacgcccac gacgcctacc tgaacgccgt cgtgggaacc gccctgatca
     7081 aaaagtaccc taagctggaa agcgagttcg tgtacggcga ctacaaggtg tacgacgtgc
     7141 ggaagatgat cgccaagagc gagcaggaaa tcggcaaggc taccgccaag tacttcttct
     7201 acagcaacat catgaacttt ttcaagaccg agattaccct ggccaacggc gagatctgga
     7261 agcggcctct gatcgagaca aacggcgaaa ccggggagat cgtgtgggat aagggccggg
     7321 attttgccac cgtgcggaaa gtgctgagca tgccccaagt gaatatcgtg aaaaagaccg
     7381 aggtgcagac aggcggcttc agcaaagagt ctatcctgcc caagaggaac agcgataagc
     7441 tgatcgccag aaagaaggac tgggacccta agaagtacgg cggcttcgac agccccaccg
     7501 tggcctattc tgtgctggtg gtggccaaag tggaaaaggg caagtccaag aaactgaaga
     7561 gtgtgaaaga gctgctgggg atcaccatca tggaaagaag cagcttcgag aagaatccca
     7621 tcgactttct ggaagccaag ggctacaaag aagtgaaaaa ggacctgatc atcaagctgc
     7681 ctaagtactc cctgttcgag ctggaaaacg gccggaagag aatgctggcc tctgccggcg
     7741 aactgcagaa gggaaacgaa ctggccctgc cctccaaata tgtgaacttc ctgtacctgg
     7801 ccagccacta tgagaagctg aagggctccc ccgaggataa tgagcagaaa cagctgtttg
     7861 tggaacagca caagcactac ctggacgaga tcatcgagca gatcagcgag ttctccaaga
     7921 gagtgatcct ggccgacgct aatctggaca aagtgctgtc cgcctacaac aagcaccggg
     7981 ataagcccat cagagagcag gccgagaata tcatccacct gtttaccctg accaatctgg
     8041 gagcccctgc cgccttcaag tactttgaca ccaccatcga ccggaagagg tacaccagca
     8101 ccaaagaggt gctggacgcc accctgatcc accagagcat caccggcctg tacgagacac
     8161 ggatcgacct gtctcagctg ggaggcgaca ccggtgctgg agacatgaga gctgccaacc
     8221 tttggccaag cccgctcatg atcaaacgct ctaagaagaa cagcctggcc ttgtccctga
     8281 cggccgacca gatggtcagt gccttgttgg atgctgagcc ccccatactc tattccgagt
     8341 atgatcctac cagacccttc agtgaagctt cgatgatggg cttactgacc aacctggcag
     8401 acagggagct ggttcacatg atcaactggg cgaagagggt gccaggcttt gtggatttga
     8461 ccctccatga tcaggtccac cttctagaat gtgcctggct agagatcctg atgattggtc
     8521 tcgtctggcg ctccatggag cacccagtga agctactgtt tgctcctaac ttgctcttgg
     8581 acaggaacca gggaaaatgt gtagagggca tggtggagat cttcgacatg ctgctggcta
     8641 catcatctcg gttccgcatg atgaatctgc agggagagga gtttgtgtgc ctcaaatcta
     8701 ttattttgct taattctgga gtgtacacat ttctgtccag caccctgaag tctctggaag
     8761 agaaggacca tatccaccga gtcctggaca agatcacaga cactttgatc cacctgatgg
     8821 ccaaggcagg cctgaccctg cagcagcagc accagcggct ggcccagctc ctcctcatcc
     8881 tctcccacat caggcacatg agtaacaaag gcatggagca tctgtacagc atgaagtgca
     8941 agaacgtggt gcccctctat gacctgctgc tggaggcggc ggacgcccac cgcctacatg
     9001 cgcccactag ccgtggaggg gcatccgtgg aggagacgga ccaaagccac ttggccactg
     9061 cgggctctac ttcatcgcat tccttgcaaa agtattacat cacgggggag gcagagggtt
     9121 tccctgccac agcttatgtc gacgagggca gaggaagtct gctaacatgc ggtgacgtcg
     9181 aggagaatcc tggcccaatg accgagtaca agcccacggt gcgcctcgcc acccgcgacg
     9241 acgtccccag ggccgtacgc accctcgccg ccgcgttcgc cgactacccc gccacgcgcc
     9301 acaccgtcga tccggaccgc cacatcgagc gggtcaccga gctgcaagaa ctcttcctca
     9361 cgcgcgtcgg gctcgacatc ggcaaggtgt gggtcgcgga cgacggcgcc gcggtggcgg
     9421 tctggaccac gccggagagc gtcgaagcgg gggcggtgtt cgccgagatc ggcccgcgca
     9481 tggccgagtt gagcggttcc cggctggccg cgcagcaaca gatggaaggc ctcctggcgc
     9541 cgcaccggcc caaggagccc gcgtggttcc tggccaccgt cggcgtctcg cccgaccacc
     9601 agggcaaggg tctgggcagc gccgtcgtgc tccccggagt ggaggcggcc gagcgcgccg
     9661 gggtgcccgc cttcctggag acctccgcgc cccacaacct ccccttctac gagcggctcg
     9721 gcttcaccgt caccgccgac gtcgaggtgc ccgaaggacc gcgcacctgg tgcatgaccc
     9781 gcaagcccgg tgccacgcgt taactaagta agcggccgcc ccggtatcga caatcaacct
     9841 ctggattaca aaatttgtga aagattgact ggtattctta actatgttgc tccttttacg
     9901 ctatgtggat acgctgcttt aatgcctttg tatcatgcgt taactaacta aacttgttta
     9961 ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca aataaagcat
    10021 ttttttcact gcattctagt tgtggtttgt ccaaactcat caatgtatct tatcatgtct
    10081 ggaattgact caaatgatgt caattagtct atcagaagct catctggtct cccttccggg
    10141 ggacaagaca tccctgttta atatttaaac agcagtgttc ccaaactggg ttcttatatc
    10201 ccttgctctg gtcaaccagg ttgcagggtt tcctgtcctc acaggaacga agtccctaaa
    10261 gaaacagtgg cagccaggtt tagccccgga attgactgga ttcctttttt agggcccatt
    10321 ggtatggctt tttccccgta tccccccagg tgtctgcagg ctcaaagagc agcgagaagc
    10381 gttcagagga aagcgatccc gtgccacctt ccccgtgccc gggctgtccc cgcacgctgc
    10441 cggctcgggg atgcgggggg agcgccggac cggagcggag ccccgggcgg ctcgctgctg
    10501 ccccctagcg ggggagggac gtaattacat ccctgggggc tttggggggg ggctgtccct
    10561 gatatctata acaagaaaat atatatataa taagttatca cgtaagtaga acatgaaata
    10621 acaatataat tatcgtatga gttaaatctt aaaagtcacg taaaagataa tcatgcgtca
    10681 ttttgactca cgcggtcgtt atagttcaaa atcagtgaca cttaccgcat tgacaagcac
    10741 gcctcacggg agctccaagc ggcgactgag atgtcctaaa tgcacagcga cggattcgcg
    10801 ctatttagaa agagagagca atatttcaag aatgcatgcg tcaattttac gcagactatc
    10861 tttctagggt taatctagct gcatcaggat catatcgtcg ggtctttttt ccggctcagt
    10921 catcgcccaa gctggcgcta tctgggcatc ggggaggaag aagcccgtgc cttttcccgc
    10981 gaggttgaag cggcatggaa agagtttgcc gaggatgact gctgctgcat tgacgttgag
    11041 cgaaaacgca cgtttaccat gatgattcgg gaaggtgtgg ccatgcacgc ctttaacggt
    11101 gaactgttcg ttcaggccac ctgggatacc agttcgtcgc ggcttttccg gacacagttc
    11161 cggatggtca gcccgaagcg catcagcaac ccgaacaata ccggcgacag ccggaactgc
    11221 cgtgccggtg tgcagattaa tgacagcggt gcggcgctgg gatattacgt cagcgaggac
    11281 gggtatcctg gctggatgcc gcagaaatgg acatggatac cccgtgagtt acccggcggg
    11341 cgcgcttggc gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa
    11401 ttccacacaa catacgagcc ggaagcataa agtgtaaagc ctggggtgcc taatgagtga
    11461 gctaactcac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt
    11521 gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct
    11581 cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat
    11641 cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga
    11701 acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt
    11761 ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt
    11821 ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc
    11881 gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa
    11941 gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct
    12001 ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta
    12061 actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg
    12121 gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc
    12181 ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta
    12241 ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg
    12301 gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt
    12361 tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg
    12421 tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta
    12481 aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg
    12541 aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg
    12601 tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc
    12661 gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg
    12721 agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg
    12781 aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag
    12841 gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat
    12901 caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc
    12961 cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc
    13021 ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa
    13081 ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac
    13141 gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt
    13201 cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc
    13261 gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa
    13321 caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca
    13381 t
//
